This 3-year R01 project is to explore a new methodology for super-resolution scanning laser ophthalmoscopy (SLO). We have demonstrated resolution-doubling in scanning laser microscopy (SLM) and optical coherence tomography (OCT) through virtually structured detection (VSD). We propose here to extend the VSD into super-resolution ophthalmoscopy for in vivo retinal imaging. Without the complexity of structured illumination microscopy (SIM), the VSD provides an easy, low-cost and phase-artifact free strategy to achieve super- resolution imaging. However, deployable application of the VSD for in vivo retinal imaging is challenged by limited frame-speed (40s per frame) of our prototype instrument. Such low speed is intolerable for in vivo retinal imaging because of within-frame blur due to eye movements. We propose here to combine rapid line-scan strategy and accurate image registration to compensate for eye movements. The first aim of this project is to construct a line-scan super-resolution SLO. Success criterion of this aim is to produce an instrument that enables super-resolution imaging at 10 ms frame-speed. In order to achieve rapid change of three-angle (0o, 30o and 60o) scanning required for super-resolution reconstruction, a galvo-dove-prism will be assembled. Preliminary line-scan imaging of frog eyes indicates that within-frame blur can be ignored at 10 ms frame- speed; while frame-by-frame movement can be corrected by accurate image registration. The second aim is to conduct both ex vivo and in vivo validation of the proposed instrument. Before in vivo experiments, a 10x objective with numeric aperture NA=0.25 will be used for super-resolution microscopy of 20 nm nanoparticles. The ex vivo experiment is designed to verify spatial resolution of the super-resolution instrument. The second aim of this project is to validate the super-resolution SLO for in vivo retinal imaging of anesthetized animals (frogs). In vivo imaging of anesthetized frog will be conducted to quantify resolution difference in super- resolution ophthalmoscopy and conventional SLO. The third aim to verify transient rod phototropism (TRP) in intact animals. We have recently demonstrated TRP in freshly isolated mouse and frog retinas. In vivo verification of the TRP will provide an optical biomarker suitable for functional mapping of rod physiology at cellular resolution. Successful endpoint of this project is to demonstrate the feasibility of super-resolution ophthalmoscopy of intact animals. In future phase of this project, the super-resolution ophthalmoscopy will be used to investigate biophysical mechanism of the TRP in normal animals, and to characterize abnormal modification of the TRP in transgenic animals with photoreceptor degeneration. Moreover, we also plan to combine adaptive optics with the proposed instrument to pursue super-resolution ophthalmoscopy of human subjects in following phase of this project.